The invention relates to an optical arrangement in the beam path of a light source suitable for fluorescence excitation, preferably in the beam path of a confocal laser scanning microscope, with at least one spectrally selective element to inject the excitation light of at least one light source in the microscope and to extract the excitation light scattered and reflected on the object or the excitation wavelength from the light coming from the object through the detection beam path.
In both conventional and confocal laser-scanning microscopy, color beam splitters with an entirely specialized transmission and reflection characteristic are used in the beam path of a light source suited for fluorescence excitation. This is predominantly a dichroic beam splitter. With an element like this, the fluorescence excitation wavelength xcexill1 or xcexill2, xcexill3 . . . , xcexilln when several lasers are used) is reflected in the illumination beam path in order to excite the fluorescence distribution in the object and then to pass through the beam path, together with the excitation light dispersed and reflected on the object, up to the color beam splitter . The excitation light with the wavelengths xcexill1, xcexill2, xcexill3, . . . , xcexilln is reflected back into the laser at the color beam splitter, specifically out of the detection beam path. The fluorescent light with the wavelengths xcexfluo1, xcexfluo2, xcexfluo3, . . . , xcexfluon passes the color beam splitter and is detected in some cases after further spectral subdivision.
Color beam splitters are typically realized by means of an interference filter and are purposively attenuated for excitation or for detection, depending on the wavelengths used. At this point it should be noted that according to the preceding description of the prior art, a wavelength-separable element that splits the light of various wavelengths on the basis of wavelength and not on the basis of polarization is understood as falling under the concept of a dichroit.
In practice the use of color beam splitters is disadvantageous to begin with in that it involves optical components that are very complex, therefore very expensive, in terms of production. It is also disadvantageous that color beam splitters have a fixed wavelength characteristic and therefore cannot be used with flexibility in terms of the wavelength of the excitation light. If the wavelength of the excitation light is changed, the color beam splitters must also be replaced, for example in an arrangement of several color beam splitters in a filter wheel. However, this is complex and therefore costly, requiring an entirely specialized adjustment of the individual color beam splitters.
The use of a color beam splitter is encumbered with the further disadvantage that certain light losses occur due to reflection, in particular light losses of fluorescent light, which is exactly what is to be detected. The spectral transmission/reflection range is rather wide for color beam splitters xcexill, xc2x120 nm) and in no way is ideally xe2x80x9csteepxe2x80x9d. Consequently, the fluorescent light from this spectral range cannot be ideally detected.
If color beam splitters are used, the number of lasers that can simultaneously inject is limited, specifically, for example, to the number of color beam splitters which are arranged in and which can be combined for a filter wheel. Typically, a maximum of three lasers is injected into the beam path. As previously explained, all color beam splitters, thus also the color beam splitters arranged in a filter wheel, must be adjusted precisely, thereby involving a substantial amount of manipulation. Alternatively, one can use suitable neutral beam splitters, which efficiently route the fluorescent light together with the excitation light scattered/reflected on the object. The losses for the laser injection here are nonetheless considerable.
For documentation of the prior art, merely refer to DE 196 27 568 A1 as an example, which shows an optical arrangement for confocal microscopy. Therefore, in concrete terms this is an arrangement for the simultaneous confocal lighting of an object plane with a multiplicity of suitable divergent light points along with accompanying imaging components and a multiplicity of pinholes for confocal contrast-rich imaging in an observation device, which can be a microscope. The injection of several light sources is made there by means of a diffractive element. Several optical splitter elements or color beam splitters are arranged in the detection beam path, resulting in a very substantial amount of added technical complexity.
As far as using active optic elements in the beam path of a laser scanning microscope is concerned, refer also to U.S. Pat. No. 4,827,125 and U.S. Pat. No. 5,410,371, said documents showing the basic use of an AOD (Acousto-Optical Deflector) and an AOTF (Acousto-Optical Tunable Filter), and specifically always with the purpose of deflecting or reducing a beam.
The object of the invention is to design and develop an optical arrangement in the beam path of a light source suitable for fluorescence excitation such that the injection of the excitation light of various excitation wavelengths is possible without having to switch or make special adjustments to the optical elements used when switching the wavelength of the excitation light. Furthermore, the number of optical elements required is to be reduced as much as possible. Finally, an ideal detection of the fluorescence light should be possible.
The inventive optical arrangement in the beam path of a light source suitable for fluorescence excitation, preferably in the beam path of a confocal laser scanning microscope, fulfills the object of the invention by means of the features of the coordinated patent claims 1 and 2. These claims describe an optical arrangement of the type in question that is characterized in that by using the spectrally selective element, excitation light of different wavelengths can be extracted or injected accordingly. Alternatively, the optical arrangement is characterized in that the spectrally selective element can be adjusted to the excitation wavelengths to be extracted.
It is recognized according to the invention that the color beam splitter previously used in the beam path of a light source suitable for fluorescence excitation, especially in the beam path of a confocal laser scanning microscope, can be replaced by a very unique spectrally selective element, specifically by a spectrally selective element that is suitable for extracting or inserting/injecting different wavelengths. This spectrally selective element is used on the one hand to inject the excitation light of at least one light source in the microscope and on the other hand to extract the excitation light scattered and reflected on the object, or the corresponding wavelengths from the light coming from the object through the detection beam path. In this respect the spectrally selective element serves a double function, both of these functions being almost mandatorily linked.
As an alternative to the capability of the spectrally selective element to extract excitation light of different wavelengths, the spectrally selective element can be adjusted to the particular excitation wavelength to be incorporated or extracted. Also in this respect based on the previously described double function, a mandatory linking is guaranteed in a simple way, namely that the excitation light can be injected in the lighting path by using the spectrally selective element and by extracting exactly the wavelength of the excitation light, namely the excitation wavelength from the light coming from the object through the detection beam path based on the adjustability provided here, so that the detection light (=fluorescent light) coming from the object remains for detection.
Advantageously, the spectrally selective elementxe2x80x94to favor the previously discussed double functionxe2x80x94can be a passive element or component. The spectrally selective element here can be configured as a transparent optical grating or as a holographic element. It is also conceivable to configure the spectrally selective element as a passive AOD (Acousto-Optical Deflector) or as a passive AOTF (Acousto-Optical Tunable Filter).
In an especially advantageous manner, specifically for the concrete realization of the adjustability of the spectrally selective element to the excitation wavelength to be extracted, the spectrally selective element can be an active component, for example an element working on an acousto-optical and/or electro-optical basis. In concrete terms this means an AOD (Acousto-Optical Deflector) or an AOTF (Acousto-Optical Tunable Filter).
Instead of the color beam splitter that is standard for the prior art, here an active spectrally selective element is used, thus for example an AOD or an AOTF. The purpose of this active component consists of injecting the excitation light of the light source or of the laser or lasers xcexill1, xcexill2, xcexill3, . . . , xcexilln into the illumination beam path and thus into the microscope in order to then activate by beam scanning the fluorescence distribution in the object. For the detection, the fluorescent light coming from the object can pass the active spectrally selective element nearly undisturbed. In the process the light scattered or reflected by the object having the excitation wavelength of the light source or of the laser or lasers from the detection beam path is largely reflected out.
For the injection of a light source or of a laser or several lasers with different wavelengths xcexill1, xcexill2, . . . , xcexilln, an AOD with corresponding frequencies v1, v2, . . . , vn can be connected, preferably simultaneously, so that the different laser beams, after passing through the AOD run coaxially with the optical axis. Regarding the use of the AOD, it is important that there a frequency vn selects a wavelength xcexilln that is deflected out of the actual beam path. The angle of deflection xcfx86 here is defined by the formula
xcfx86=xcexilln vn/2f
f being the expansion speed of the sound wave in the AOD. The fluorescent light to be detected with a spectral distribution around the wavelengths xcexfluo1, xcexfluo2, . . . , xcexfluon together with the excitation light scattered or reflected on the object with the wavelengths xcexill1, xcexill2, . . . , xcexilln then passes through the AOD in the reverse direction. Nevertheless, the excitation light with the wavelengths xcexill1, xcexill2, . . . , xcexilln is deflected out of the detection beam path in the direction of the laser according to the reversibility of the light path based of the specific setting of the AOD (1st order). Thus, the xe2x80x9cspectrally remnantxe2x80x9d fluorescent light around the wavelengths xcexfluo1, xcexfluo2, . . . , xcexfluon can be detected in an improved way, compared with a standard color beam splitter (0-order). In this way the adjustment of the injection of different lasers can in any event be made more simply than in the prior art (there using standard color beam splitters in a filter wheel).
In an additional advantageous way, connecting additional AOTF""s could selectively regulate the individual wavelengths in their power after the merging of the beams.
To insert a laser light source with various wavelengths xcexfill1, xcexfill2, . . . , xcexfilln, an AOTF with corresponding frequencies v1, v2, . . . , vn can be simultaneously connected so that the different wavelengths vary in their excitation power and can be optimized according to application. The supply of laser light can be made using a fiber optical waveguide.
In any case, the light source or laser is coaxially injected from the direction of the 1st order of the crystal. The fluorescent light to be detected with a spectral distribution around the wavelengths xcexfluo1, xcexfluo2, . . . , xcexfluon, together with the excitation light with wavelengths xcexfill1, xcexfill2, . . . , xcexfiln scattered or reflected on the object then pass through the AOTF in the reverse direction. According to the reversibility of the light path, the excitation light with the wavelengths xcexfill1, xcexfill2, . . . , xcexfiln, are deflected out of the detection path in the direction of the light source or laser because of the specific setting of the AOTF. Thus, the xe2x80x9cspectrally remnantxe2x80x9d fluorescent light around the wavelengths xcexfluo1, xcexfluo2, . . . , xcexfluon can be detected (0-order) inxe2x80x94compared to the standard color beam splitterxe2x80x94an improved way.
Using either an AOD or AOTF or even a transparent grating, the fluorescent light, after passing through the particular active element, will spectrally fan itself out based on the dispersion that occurs. In this respect, it is advantageous to connect one or more corresponding xe2x80x9cinversexe2x80x9d elements downstream so that the undesired spectral fanning out is made to go in reverse again. It is also conceivable to connect additional optical elements upstream or downstream from the element in question (AOD, AOTF or transparent grating) for focusing or for extraction of undesired beam portions. The detection beam reunited in this way can then be spectrally dissected and depicted on the different detectors in a conventional manner using downstream color beam splitters.
Fundamentally, an arrangement in the sense of a xe2x80x9cmulti-band detectorxe2x80x9d is conceivable. Refer to the patent application DE 43 30 347.1-42, the content of which is expressly consulted here and in this respect is assumed to be known in the art. The excitation pinhole, this being identical to the detection pinhole, is arranged between the scan unit and the AOD, the transparent grating (with several light sources or lasers of several wavelengths) or the AOTF (with one light source or one laser with various wavelengths). In an advantageous way, the characteristic of the crystal here of spectrally fanning out the light beam of the 0-order by means of the prism effect is used for detection. The dispersive element of the multi-band detector is combined here with the color beam splitter into one component so that all additional color beam splitters connected downstream from the detection beam path and encumbered with additional losses in the intensity of the fluorescence are eliminated.
In a very unique way, the previously discussed technique in combination with a variable laser light source that is variable in wave lengthxe2x80x94e.g. dye laser, OPO (optically parameterized oscillator), electron beam collision light sourcexe2x80x94can enable exceptionally flexible fluorescence microscopy applications. The setting or control of the excitation wavelength can be coupled directly to the drive unit of one of the previously described spectrally selective elements so that only this excitation wavelength is injected and in turn only this wavelength is extracted from the detection beam path. The coupling or mandatory coupling of the light source to the beam-splitting element can be accomplished either manually or automatically or perhaps according a pre-determinable specification, whereby this possibility must be adapted to the current requirement profile. For example, after each scanned focal plane, the excitation wavelength and the beam splitter can be changed in a suitable way. In this way multi-colored fluorescence objects can be detected. A conversion by lines is also conceivable.
The advantages of the concept according to the invention can be summarized in conjunction with an advantageous embodiment as follows:
The spectrally selective elements are xe2x80x9ctransparentxe2x80x9d for all wavelengths except for the selected excitation wavelengths xcexill1, xcexill2, . . . , xcexilln. The xe2x80x9cspectral lossxe2x80x9d is minimal, since only the selected spectral range of typically xcexilln xc2x12 nm is deflected by the spectrally selective element. As a result, the spectral range for the detection is expanded. As a result almost any of the many different wavelength ranges can be simultaneously injected and used. The spectrally xe2x80x9clost fluorescent intensityxe2x80x9d that is caused by the spectrally selective elements is less than for standard color beam splitters. In other words, there are reduced losses in intensity in the range in question. The active spectrally selective elements can be flexibly adjusted so that in principle many light sources or lasers with various wavelengths can also be injected simultaneously into the microscope. This enables the improved application with multi-color FISH (Fluorescence-In-Situ Hybridization). Consequently, there is still only a limitation of the spectral splitting of the fluorescent light, for example, due to xe2x80x9ccross-talkxe2x80x9d. Standard barrier filters can be completely omitted so that additional losses of fluorescent light in the detection are prevented.
Finally, it is also conceivable that another active holographic element be connected downstream from the spectrally selective element and the task of the beam scanner be performed in the process. Both elements can be combined into a single component.
In principle different light sources can be used as long as they are suited for fluorescent excitation. The following come to mind as examples: a white light source, a light source for using an optic parameterized oscillator, an electron beam collision light source or a laser light source, the latter capable of being varied through wavelength variation. Laser light sources with different wavelengths or a light source comprising several lasers can be used.